Previous results suggest that Ehrlich ascites tumor cell NaK-ATPase, but not NaK-ATPase isolated from normal cells, is phosphorylated on the enzyme's beta subunits. As part of our effort to determine if this observation indicates a protein kinase-\and protein phosphatase-dependent phosphorylation/dephosphorylation regulatory cycle of NaK-ATPase which is defective in the tumor cell, we have examined the ascites cell in the hope of finding phosphatase activity. As a result, we reported the presence of a soluble, Mg2+-\or Mn2+-dependent p-nitrophenylphosphatase activity in Ehrlich ascites tumor cell homogenates. The crude homogenate was fractionated over Sephadex G-150 gel filtration and DEAE-Sephacel anion exchange columns, and two partially purified p-nitrophenylphosphatase activities were resolved. The most active fraction, Peak I, was characterized and found to be similar to phosphotyrosyl-protein phosphatases characterized elsewhere in that it has optimal activity at neutral pH, it is inhibited by phosphate, Zn2+, and vanadate, but is notinhibited by levamisole. However, Peak I differs from phosphotyrosyl-protein phosphatases in that Mg2+ or Mn2+ is required for activity, fluoride is an inhibitor, and pyrophospate is not inhibitory. Inhibition by the phosphorylated compounds phosphotyrosine, phosphoserine, phosphothreonine, ATP, CTP, GTP, ITP, NADP, fructose-6-phosphate, glycose-1-phosphate, galactose-1-phosphate, 2-phosphogluconic acid, and 6-phosphogluconic acid was also observed. Ehrlich ascites tumor cell p-nitrophenylphosphatase is shown to be sensitive to inactivation by trypsin, N-ethylmaleimide, or heat treatments. (E)